Filamentous fungi, such as Aspergillus niger, are well known for their industrial applications in protein and chemical productions. They are used to produce a wide variety of products ranging from human therapeutics, glycosyl hydrolases to specialty chemicals (Punt et al., Trends Biotechnol 20(5):200-206, 2002; Schuster et al., Appl Microbiol Biotechnol 59(4-5):426-435, 2002; Gerngross, Nat Biotechnol 22(11):1409-1414, 2004; Nevalainen et al., Trends Biotechnol 23(9):468-474, 2005; Sauer et al., Trends Biotechnol. 26(2):100-8, 2008; Magnuson and Lasure (2004). “Organic acid production by filamentous fungi.” Advances in fungal biotechnology for industry, agriculture, and medicine, pages 307-340). Some of industrial A. niger strains are capable of growing on solutions of glucose or sucrose in excess of 20% (w/v) and converting approximately 90% of the supplied carbohydrate to citric acid. These remarkable properties are the reason that A. niger has been used to produce citric acid for more 80 years and is currently the primary source of commercial citric acid production (Magnuson and Lasure (2004). “Organic acid production by filamentous fungi.” Advances in fungal biotechnology for industry, agriculture, and medicine, pages 307-340).
The maximum product output in fermentation processes is the result of optimal metabolic pathways and cellular formation, which are influenced by endogenous and exogenous factors. Cellular metabolisms are tightly controlled and highly interconnected, and are regulated spatially and temporally at different levels, such as transcription, post-transcription, translation, and post-translation. Therefore, different approaches have been explored to understand the regulatory mechanisms of metabolic processes and cellular formation for maximizing the product output in filamentous fungi. For example, comparative genomics was used to examine citric-acid-producing versus enzyme-producing A. niger strains (Andersen et al., Genome Res. 21(6): 885-97, 2011), proteomics was used to examine filamentous fungi related to enzymes or organic acid production (de Oliveira and de Graaff, Appl. Microbiol. Biotechnol. 89(2): 225-37, 2011), or combination of both genomics and proteomics were used to examine enzyme production (Jacobs et al., Fungal Genetics and Biology 46(1, Supplement):S141-S152, 2009). Although these studies examined the potential involvement of selected genes and proteins in optimizing production of organic acids or proteins in filamentous fungi, methods for altering the complex post-translation modifications (such as N-glycosylation of cellular proteins) for signal transduction, cellular formation and metabolism at different growth and development stages, which may affect product output, have not been examined.
Protein glycosylation is a ubiquitous and structurally diverse form of post translation modification, which occurs at all domains of life. More than two-thirds of eukaryotic proteins are predicted to be glycosylated (Apweiler et al., Biochim Biophys Acta 1473(1):4-8, 1999). N- and O-linked protein glycosylation are common types of protein glycosylation, occurring mainly on the asparagine (N) and serine/threonine (S/T) residues, respectively. N-linked glycosylation has been implicated in many biochemical and cellular processes, including protein secretion, stability and translocation, maintenance of cell structure, receptor-ligand interactions and cell signaling, cell-cell recognition, pathogen infection, and host defense in various organisms (Haltiwanger and Lowe, Ann. Rev. Biochem.73(1):491-537, 2004; Dellaporta et al., Plant Mol. Biol. Reporter 1(4):19-21, 1983; Nam et al., Biotech. Bioengineer. 100(6): 1178-1192, 2008; Trombetta and Parodi, Ann. Rev. Cell Dev. Biol. 19(1):649-676, 2003; Tsang et al., Fungal Genetics and Biology 46(1): S153-S160, 2009; Pang et al., Science, 333(6050):1761-4, 2011).
N-glycosylation is highly complex and has been extensively studied in mammalian systems (Yan and Lennarz, J. Biol. Chem. 280(5):3121, 2005; Silberstein and Gilmore, FASEB J. 10(8): 849, 1996; Kornfeld and Kornfeld, Annu. Rev. Biochem. 54:631-664, 2005; Kim et al., PLoS ONE 4(10): e7317, 2009, 2009) and yeast (Kukuruzinska et al., Annu. Rev. Biochem. 56(1):915-944, 1987). The protein N-glycosylation pathways in filamentous fungi have also been identified (Deshpande et al., Glycobiology 18(8):626-637, 2008; Geysens et al., Fungal Genetics and Biology 46(1, Supplement): S121-S140, 2009) on the basis of the known genomic sequences. Several genes involved in N-glycosylation have been studied in filamentous fungi (Kotz et al., PLoS ONE 5(12):e15729, 2010; Kainz et al., Appl Environ Microbiol 74(4):1076-86, 2008; Maras et al., J. Biotechnol. 77(2-3):255-63, 2000; Maddi and Free, Eukaryot Cell 9(11):1766-75, 2010; Bowman et al., Eukaryotic Cell 5(3):587-600, 2006). In these studies, the effects of gene deletion on N-linked glycan patterns formation, the cell wall formation, overall protein secretion and/or the phenotypic changes were demonstrated.
Alg3 is localized in the ER and catalyzes the initial transfer of a mannose residue from dolichol pyrophosphate-mannose to lipid-linked Man5GlcNAc2-PP-Dol on the ER luminal side. It is involved in the early N-glycan synthesis in eukaryotes for the assembly of a Glc3Man9GlcNAc2 core oligosaccharide that is linked to the lipid carrier dolichol pyrophosphate. The Alg3 gene and its functions have been identified and studied in S. cerevisiae, P. pastoris, T. brucei, A. thaliana, and human (Aebi et al., Glycobiol. 6(4):439-444, 1996; Korner et al., EMBO J. 18(23): 6816-6822, 1999; Davidson et al., Glycobiology 14(5):399-407, 2004; Manthri et al., Glycobiol. 18(5):367-83, 2008; Kajiura et al., Glycobiol. 20(6):736-51, 2010). In these studies, the Alg3 mutants exhibited a unique structural profile in the glycoproteins, such as Man3GlcNAc2, Man4GlcNAc2, Man5GlcNAc2, GlcMan5GlcNAc2, and Glc3Man5GlcNAc2, which affected the overall N-glycosylation by incomplete utilization of N-linked glycosites in glycoproteins. No obvious growth phenotype was observed in those Alg3Δ mutants of S. cerevisiae, P. pastoris, T. brucei, and plant except that the Alg3 defect in human caused severe diseases such as profound psychomotor delay, optic atrophy, acquired microcephaly, iris olobomas and hypsarrhythmia (Stibler et al., Neuropediatrics 26(5): 235-7, 1995; Sun et al., J. Clin. Endocrinol. Metab. 90(7):4371-5, 2005; Schollen et al., Eur. J. Med. Genet. 48(2):153-158, 2005, Kranz et al., Am. J. Med. Genet. 143A(13):1414-20, 2007; Denecke et al., Pediatr. Res. 58(2):248-53, 2005).
LaeA, a global regulator gene for the secondary metabolism, was first identified in A. nidulans through complementing the aflR deficient mutants (Bok and Keller, Eukaryot Cell 3:527-535, 2004). Deletion of LaeA gene inhibits the expression of secondary metabolic gene clusters, such as sterigmatocystin, penicillin, and lovastin, but has no effect on spore production in A. nidulans. The LaeA that was confirmed as a nuclear protein and a putative methyltransferase does not involve in gene clusters for nutrient utilization (Bok et al., Mol Microbiol 61:1636-45, 2006). Furthermore, the role of LaeA in secondary metabolism was confirmed in Aspergillus flavus and Aspergillus oryzae (Kale et al., Fungal Genet. Biol. 45:1422-9, 2008; Oda et al., Biosci Biotechnol Biochem 75:1832-4, 2011). Evidence indicates that LaeA reverses gene repression at the level of the heterochromatin state (Reyes-Dominguez et al., Molecular Microbiology 76:1376-86, 2010). LaeA is a component of the heterotrimeric VeA/VelB/LaeA protein complex (Bayram et al., Science Signalling 320:1504, 2008), which involves in the acetylation signal transduction for secondary metabolite production in A. nidulans (Soukup et al., Mol. Microbiol., 86(2):314-30, 2012). The veA/VelB/LaeA complex may coordinately respond to environmental cues (Ramamoorthy et al., Mol. Microbiol., 85(4):795-814, 2012) and has a role in fungal morphology (Calvo, Fungal Genetics and Biology 45:1053-61, 2008). LaeA may direct the formation of the VelB-VosA and VelB-VelA-LaeA complexes, control veA modification and protein levels, and be involved in light regulation of growth and development (Bayram et al., PLoS genetics 6: e1001226, 2010).